Organic chemicals such as organic acids, esters, and polyols can be used to synthesize plastic materials and other products. To meet the increasing demand for organic chemicals, more efficient and cost-effective production methods are being developed which utilize raw materials based on carbohydrates rather than hydrocarbons. For example, certain bacteria have been used to produce large quantities of lactic acid used in the production of polylactic acid.
3-hydroxypropionic acid (3-HP) is an organic acid. Several chemical synthesis routes have been described to produce 3-HP, and biocatalytic routes have also been disclosed (WO 01/16346 to Suthers et al.). 3-HP has utility for specialty synthesis and can be converted to commercially important intermediates by known methods in the chemical industry, e.g., acrylic acid by dehydration, malonic acid by oxidation, esters by esterification reactions with alcohols, and 1,3-propanediol by reduction.
The compound 3-HP can be produced biocatalytically from PEP or pyruvate, through a key beta-alanine intermediate (FIG. 1). Beta-alanine can be synthesized in cells from carnosine, beta-alanyl arginine, beta-alanyl lysine, uracil via 5,6-dihydrouracil and N-carbamoyl-beta-alanine, N-acetyl-beta-alanine, anserine, or aspartate. However, these routes are commercially unviable because they require rare precursors or starting compounds that are more valuable than 3-HP. Therefore, production of 3-HP using biocatalytic routes would be more efficient if alpha-alanine could be converted to beta-alanine directly (FIG. 1). Unfortunately, a naturally occurring enzyme that inter-converts alpha-alanine to beta-alanine has not yet been identified. It would be advantageous if enzymatic activities that carry out the conversion of alpha-alanine to beta-alanine were identified, such as an alanine 2,3-aminomutase. Accordingly, it is one object of the present invention to identify enzymes with improved alanine 2-3-aminomutase activity.
Lysine 2,3-aminomutase (KAM), which catalyzes the anaerobic interconversion of lysine to beta-lysine, was first described by Barker in Clostridium SB4 (now C. subterminale) catalyzing the first step in the fermentation of lysine. KAM has been purified from C. subterminale, the gene cloned and expressed in E. coli. See e.g., U.S. Pat. No. 6,248,874, which issued on Jun. 19, 2001 to Frey et al., the whole of which is hereby incorporated herein by reference. The specific activity of purified KAM from C. subterminale SB4 cells has been reported as 3040 units/mg (Lieder et. al., Biochemistry 37:2578 (1998)), where a unit is defined as μmoles lysine/min. The corresponding purified recombinantly produced KAM had equivalent enzyme activity (34.5±1.6 μmoles lysine/min/mg protein). See U.S. Patent Application Publication No. 2003/0113882 A1, which published on Jun. 19, 2003 to Frey et al., the whole of which is incorporated herein by reference.
Based upon the sequence of the KAM from C. subterminale, KAM genes have been annotated in the genomes of other organisms. However, in most cases, the enzymatic activities of the polypeptides encoded by these genes have not been confirmed. Exceptions are the B. subtilis gene (Chen, D., Ruzicka, F. J., and Frey, P. A. (2000) Biochem. J. 348:539-549)), and the Porphyromonas gingivalis and F. nucleatum genes. The B. subtilis KAM, encoded by the yodO gene, is more resistant to O2 than the C. subterminale KAM, but it is markedly less active. As reported by Frey, the B. subtilis KAM has a specific activity of only 0.62 U/mg.
C. subterminale SB4 KAM has been reported to have some cross-reactivity with L-alanine, converting it into beta-alanine. See U.S. Patent Application Publication No. 2003/0113882 A1. WO 03/062173 and WO 02/42418 disclose the first reports of AAM activity based upon modification of kam genes. In these applications, the synthetic aam genes had AAM activity as detected by the complementation of a ΔpanD E. coli strain. However, because alanine is not the natural substrate for this enzyme, the activity for this conversion is substantially less than the activity for conversion of lysine—its natural substrate. The AAM activity of a variant of B. subtilis KAM that also had AAM activity at approximately 0.001 U/mg. It is an object of the present invention to provide polynucleotides encoding a polypeptide having substantially enhanced AAM activity over that found in the wild-type enzymes.